Probing nuclear localization signal-importin alpha binding equilibria in living cells

Area of Science:

• Cell biology
• Molecular mechanisms of nuclear transport
• Biophysics of protein-nucleic acid interactions

Background:

• Nuclear protein import is essential for eukaryotic cell function.
• This process is mediated by nuclear localization signals (NLSs) and import receptors like importin alpha (Impalpha).
• Understanding the quantitative details of NLS-Impalpha recognition in vivo is crucial.

Purpose of the Study:

• To quantitatively characterize the physicochemical details of NLS recognition by Impalpha in living cells.
• To investigate the regulation of nuclear import rates based on NLS-Impalpha interactions.
• To explore the role of the Impalpha autoinhibition mechanism in nuclear import.

Main Methods:

• Fluorescence recovery after photobleaching (FRAP) for quantifying nuclear import saturability and binding affinity.
• Fluorescence lifetime imaging microscopy (FLIM) to monitor NLS-Impalpha interactions and measure dissociation constants (K(D)) in situ.
• Kinetic and thermodynamic analyses of in vivo data.

Main Results:

• Quantitative determination of NLS-Impalpha binding affinity and nuclear import saturability in living cells.
• Direct measurement of NLS-Impalpha interaction K(D) within the cellular environment.
• Demonstration that Impalpha subsaturation with NLS cargo, not in vitro predicted saturation, governs nuclear import rates.
• Evidence for regulation of nuclear import by the Impalpha autoinhibition mechanism, with an estimated autoinhibition energy.

Conclusions:

• Nuclear import rates in living cells are regulated by the subsaturation of Impalpha with NLS cargo, contrary to in vitro predictions.
• The Impalpha autoinhibition mechanism plays a role in regulating nuclear import.
• This study provides the first estimate of Impalpha autoinhibition energy in vivo.